Identification of new human gaba transporter

ABSTRACT

Human GABA transporter2 like protein polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing human GABA transporter2 like protein polypeptides and polynucleotides in diagnostic assays.

FIELD OF THE INVENTION

This invention relates to newly identified polypeptides andpolynucleotides, encoding such polypeptides sometimes hereinafterreferred to as human GABA transporter2 like protein, to their use indiagnosis and in identifying compounds that may be agonists, antagoniststhat are potentially useful in therapy, and to production of suchpolypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

The drug discovery process is currently undergoing a fundamentalrevolution as it embraces “functional genomics”, that is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperceding earlier approaches based on “positional cloning”. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

Functional genomics relies heavily on high-throughput DNA sequencingtechnologies and the various tools of bioinformatics to identify genesequences of potential interest from the many molecular biologydatabases now available. There is a continuing need to identify andcharacterise further genes and their related polypeptides/proteins, astargets for drug discovery.

The amino acid γ-aminobutyric acid (GABA) is a dominant inhibitoryneurotransmitter in brain and was found widely distributed throughoutthe nervous system. For the mouse homolog of the novel GABA transportera developmentally regulated expression was determined. The mRNA of themouse homolog NTG2 is abundant in neonatal brain. The NTG2 proteintransports γ-aminobutyric acid, β-alanine and taurine (Liu QR et al(1993) J. Biol. Chem 268 (3), 2106–2112). The γ-aminobutyric acid (GABA)is a neurotransmitter involved in various central nervous systemdiseases.

Diseases to be treated including but are not limited to schizophrenia,epilepsy, depression, learning disorders, cognitive disorders,neurodegenerative diseases, multiple sclerosis, dementia, Alzheimersdisease, Parkinsons disease, hyperactivity, anxiety disorder, sleepingdisorder, alcoholism, muscular disorders (e.g. tremor), pain, headache,and migraine.

SUMMARY OF THE INVENTION

The present invention relates to GAT2, human GABA transporter2 likeprotein, in particular GAT2, human GABA transporter2 like proteinpolypeptides and GAT2, human GABA transporter2 like proteinpolynucleotides, recombinant materials and methods for their production.Such polypeptides and polynucleotides are of interest in relation tomethods of treatment of certain diseases, including, but not limited tothe diseases listed above, hereinafter referred to as “diseases of theinvention”. In a further aspect, the invention relates to methods foridentifying agonists and antagonists (e.g., inhibitors) using thematerials provided by the invention, and treating conditions associatedwith GAT2, human GABA transporter2 like protein imbalance with theidentified compounds. In a still further aspect, the invention relatesto diagnostic assays for detecting diseases associated withinappropriate GAT2, human GABA transporter2 like protein activity orlevels.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to GAT2, human GABAtransporter2 like protein polypeptides. Such polypeptides include:

(a) a polypeptide encoded by a polynucleotide comprising the sequence ofSEQ ID NO:1;

(b) a polypeptide comprising a polypeptide sequence having at least 95%,96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ IDNO:2;

(c) a polypeptide comprising the polypeptide sequence of SEQ ID NO:2;

(d) a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity tothe polypeptide sequence of SEQ ID NO:2;

(e) the polypeptide sequence of SEQ ID NO:2; and

(f) a polypeptide having or comprising a polypeptide sequence that hasan Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to thepolypeptide sequence of SEQ ID NO:2;

(g) fragments and variants of such polypeptides in (a) to (f).

Polypeptides of the present invention are believed to be members of theSodium- and chloride-dependent GABA transporter family of polypeptides.They are therefore of interest because they terminate the action of GABAby sodium-dependent reuptake into presynaptic terminals and can alsotransport beta-alanine and taurine.

The biological properties of the GAT2, human GABA transporter2 likeprotein are hereinafter referred to as “biological activity of GAT2,human GABA transporter2 like protein” or “GAT2, human GABA transporter2like protein activity”. Preferably, a polypeptide of the presentinvention exhibits at least one biological activity of GAT2, human GABAtransporter2 like protein.

Polypeptides of the present invention also includes variants of theaforementioned polypeptides, including all allelic forms and splicevariants. Such polypeptides vary from the reference polypeptide byinsertions, deletions, and substitutions that may be conservative ornon-conservative, or any combination thereof. Particularly preferredvariants are those in which several, for instance from 50 to 30, from 30to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to1 or 1 amino acids are inserted, substituted, or deleted, in anycombination.

Preferred fragments of polypeptides of the present invention include apolypeptide comprising an amino acid sequence having at least 30, 50 or100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2,or a polypeptide comprising an amino acid sequence having at least 30,50 or 100 contiguous amino acids truncated or deleted from the aminoacid sequence of SEQ ID NO: 2. Preferred fragments are biologicallyactive fragments that mediate the biological activity of GAT2, humanGABA transporter2 like protein, including those with a similar activityor an improved-activity, or with a decreased undesirable activity. Alsopreferred are those fragments that are antigenic or immunogenic in ananimal, especially in a human.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention. Thepolypeptides of the present invention may be in the form of the “mature”protein or may be a part of a larger protein such as a precursor or afusion protein. It is often advantageous to include an additional aminoacid sequence that contains secretory or leader sequences,pro-sequences, sequences that aid in purification, for instance multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

Polypeptides of the present invention can be prepared in any suitablemanner, for instance by isolation form naturally occuring sources, fromgenetically engineered host cells comprising expression systems (videinfra) or by chemical synthesis, using for instance automated peptidesynthesisers, or a combination of such methods. Means for preparing suchpolypeptides are well understood in the art.

In a further aspect, the present invention relates to GAT2, human GABAtransporter2 like protein polynucleotides. Such polynucleotides include:

(a) a polynucleotide comprising a polynucleotide sequence having atleast 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide sequenceof SEQ ID NO:1;

(b) a polynucleotide comprising the polynucleotide of SEQ ID NO:1;

(c) a polynucleotide having at least 95%, 96%, 97%, 98%, or 99% identityto the polynucleotide of SEQ ID NO:1;

(d) the polynucleotide of SEQ ID NO:1;

(e) a polynucleotide comprising a polynucleotide sequence encoding apolypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identityto the polypeptide sequence of SEQ ID NO:2;

(f) a polynucleotide comprising a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2;

(g) a polynucleotide having a polynucleotide sequence encoding apolypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identityto the polypeptide sequence of SEQ ID NO:2;

(h) a polynucleotide encoding the polypeptide of SEQ ID NO:2;

(i) a polynucleotide having or comprising a polynucleotide sequence thathas an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to thepolynucleotide sequence of SEQ ID NO:1;

(j) a polynucleotide having or comprising a polynucleotide sequenceencoding a polypeptide sequence that has an Identity Index of 0.95,0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ IDNO:2; and

polynucleotides that are fragments and variants of the above mentionedpolynucleotides or that are complementary to above mentionedpolynucleotides, over the entire length thereof.

Preferred variants of polynucleotides of the present invention include apolynucleotide comprising an nucleotide sequence having at least 15, 30,50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or apolynucleotide comprising an sequence having at least 30, 50 or 100contiguous nucleotides truncated or deleted from the sequence of SEQ IDNO: 1.

Preferred variants of polynucleotides of the present invention includesplice variants, allelic variants, and polymorphisms, includingpolynucleotides having one or more single nucleotide polymorphisms(SNPs).

Polynucleotides of the present invention also include polynucleotidesencoding polypeptide variants that comprise the amino acid sequence ofSEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1or 1 amino acid residues are substituted, deleted or added, in anycombination.

In a further aspect, the present invention provides polynucleotides thatare RNA transcripts of the DNA sequences of the present invention.Accordingly, there is provided an RNA polynucleotide that:

-   -   (a) comprises an RNA transcript of the DNA sequence encoding the        polypeptide of SEQ ID NO:2;    -   (b) is the RNA transcript of the DNA sequence encoding the        polypeptide of SEQ ID NO:2;    -   (c) comprises an RNA transcript of the DNA sequence of SEQ ID        NO:1; or    -   (d) is the RNA transcript of the DNA sequence of SEQ ID NO:1;    -   and RNA polynucleotides that are complementary thereto.

The polynucleotide sequence of SEQ ID NO:1 shows homology with MouseSodium- and chloride-dependent GABA transporter 2 (GAT3) (Other namesare SLCA13, GABT2 or GAT-2). The sequence of mouse GAT3 was placed underthe accession number P31649 in Swiss-prot by Liu QR et al (J. Biol.Chem. 268: 2106–2112 (1993). The polynucleotide sequence of SEQ ID NO:1is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2. Thepolynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may beidentical to the polypeptide encoding sequence of SEQ ID NO:1 or it maybe a sequence other than SEQ ID NO:1, which, as a result of theredundancy (degeneracy) of the genetic code, also encodes thepolypeptide of SEQ ID NO:2. The polypeptide of the SEQ ID NO:2 isrelated to other proteins of the Sodium- and chloride-dependent GABAtransporter family, having homology and/or structural similarity withMouse Sodium- and chloride-dependent GABA transporter 2 (GAT3) (Othernames are SLCA13, GABT2 or GAT-2). The sequence of mouse GAT3 was placedunder the accession number P31649 in Swiss-prot by Liu Q R et al (J.Biol. Chem. 268: 2106–2112 (1993).

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides and polynucleotides of the present invention haveat least one GAT2, human GABA transporter2 like protein activity.

Polynucleotides of the present invention may be obtained using standardcloning and screening techniques from a cDNA library derived from mRNAin cells of human brain, (see for instance, Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)). Polynucleotides of theinvention can also be obtained from natural sources such as genomic DNAlibraries or can be synthesized using well known and commerciallyavailable techniques.

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself, or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence that facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821–824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed;non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Polynucleotides that are identical, or have sufficient identity to apolynucleotide sequence of SEQ ID NO:1, may be used as hybridizationprobes for cDNA and genomic DNA or as primers for a nucleic acidamplification reaction (for instance, PCR). Such probes and primers maybe used to isolate full-length cDNAs and genomic clones encodingpolypeptides of the present invention and to isolate cDNA and genomicclones of other genes (including genes encoding paralogs from humansources and orthologs and paralogs from species other than human) thathave a high sequence similarity to SEQ ID NO:1, typically at least 95%identity. Preferred probes and primers will generally comprise at least15 nucleotides, preferably, at least 30 nucleotides and may have atleast 50, if not at least 100 nucleotides. Particularly preferred probeswill have between 30 and 50 nucleotides. Particularly preferred primerswill have between 20 and 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess comprising the steps of screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Thus the present invention alsoincludes isolated polynucleotides, preferably with a nucleotide sequenceof at least 100, obtained by screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO:1 or a fragment thereof, preferably of at least 15 nucleotides.

The skilled artisan will appreciate that, in many cases, an isolatedcDNA sequence will be incomplete, in that the region coding for thepolypeptide does not extend all the way through to the 5′ terminus. Thisis a consequence of reverse transcriptase, an enzyme with inherently low“processivity” (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during first strand cDNAsynthesis.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998–90021988). Recent modifications of the technique, exemplified by theMARATHON (trade mark) technology (Clontech Laboratories Inc.) forexample, have significantly simplified the search for longer cDNAs. Inthe MARATHON (trade mark) technology, cDNAs have been prepared from mRNAextracted from a chosen tissue and an ‘adaptor’ sequence ligated ontoeach end. Nucleic acid amplification (PCR) is then carried out toamplify the “missing” 5′ end of the cDNA using a combination of genespecific and adaptor specific oligonucleotide primers. The POR reactionis then repeated using ‘nested’ primers, that is, primers designed toanneal within the amplified product (typically an adaptor specificprimer that anneals further 3′ in the adaptor sequence and a genespecific primer that anneals further 5′ in the known gene sequence). Theproducts of this reaction can then be analysed by DNA sequencing and afull-length cDNA constructed either by joining the product directly tothe existing cDNA to give a complete sequence, or carrying out aseparate full-length PCR using the new sequence information for thedesign of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems comprising apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Polynucleotides may be introduced into hostcells by methods described in many standard laboratory manuals, such asDavis et al., Basic Methods in Molecular Biology (1986) and Sambrook etal.(ibid). Preferred methods of introducing polynucleotides into hostcells include, for instance, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector that is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate polynucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., (ibid). Appropriatesecretion signals may be incorporated into the desired polypeptide toallow secretion of the translated protein into the lumen of theendoplasmic reticulum, the periplasmic space or the extracellularenvironment. These signals may be endogenous to the polypeptide or theymay be heterologous signals.

If a polypeptide of the present invention is to be expressed for use inscreening assays, it is generally preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocelluiose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured duringintracellular synthesis, isolation and/or purification.

Polynucleotides of the present invention may be used as diagnosticreagents, through detecting mutations in the associated gene. Detectionof a mutated form of the gene characterised by the polynucleotide of SEQID NO:1 in the cDNA or genomic sequence and which is associated with adysfunction will provide a diagnostic tool that can add to, or define, adiagnosis of a disease, or susceptibility to a disease, which resultsfrom under-expression, over-expression or altered spatial or temporalexpression of the gene. Individuals carrying mutations in the gene maybe detected at the DNA level by a variety of techniques well known inthe art.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or it may be amplifiedenzymatically by using PCR, preferably RT-PCR, or other amplificationtechniques prior to analysis. RNA or cDNA may also be used in similarfashion. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to labeledGAT2, human GABA transporter2 like protein nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence difference may also be detected by alterations in theelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing (see, for instance, Myerset al., Science (1985) 230:1242). Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397–4401).

An array of cligonucleotides probes comprising GAT2, human GABAtransporter2 like protein polynucleotide sequence or fragments thereofcan be constructed to conduct efficient screening of e.g., geneticmutations. Such arrays are preferably high density arrays or grids.Array technology methods are well known and have general applicabilityand can be used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability,see, for example, M. Chee et al., Science, 274, 610–613 (1996) and otherreferences cited therein.

Detection of abnormally decreased or increased levels of polypeptide ormRNA expression may also be used for diagnosing or determiningsusceptibility of a subject to a disease of the invention. Decreased orincreased expression can be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides,such as, for example, nucleic acid amplification, for instance PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods. Assay techniques that can be used to determine levels of aprotein, such as a polypeptide of the present invention, in a samplederived from a host are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagonostickit comprising:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence of SEQ ID NO: 1, or a fragment or an RNA transcript thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO:2 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularlydiseases of the invention, amongst others.

The polynucleotide sequences of the present invention are valuable forchromosome localisation studies. The sequence is specifically targetedto, and can hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (co-inheritance of physically adjacent genes). Precisehuman chromosomal localisations for a genomic sequence (gene fragmentetc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M.Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) Amethod for constructing radiation hybrid maps of whole genomes, NatureGenetics 7, 22–28). A number of RH panels are available from ResearchGenetics (Huntsville, Ala., USA) e.g. the GeneBridge4 RH panel (Hum MolGenet 1996 March;5(3):339–46 A radiation hybrid map of the human genome.Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D,Muselet D, Prud'Homme J F, Dib C, Auffray C, Morissette J, WeissenbachJ, Goodfellow P N). To determine the chromosomal location of a geneusing this panel, 93 PCRs are performed using primers designed from thegene of interest on RH DNAs. Each of these DNAs contains random humangenomic fragments maintained in a hamster background (human/hamsterhybrid cell lines). These PCRs result in 93 scores indicating thepresence or absence of the PCR product of the gene of interest. Thesescores are compared with scores created using PCR products from genomicsequences of known location. This comparison is conducted on the worldwide web address penome.wi.mit.edu/.

The polynucleotide sequences of the present invention are also valuabletools for tissue expression studies. Such studies allow thedetermination of expression patterns of polynucleotides of the presentinvention which may give an indication as to the expression patterns ofthe encoded polypeptides in tissues, by detecting the mRNAs that encodethem. The techniques used are well known in the art and include in situhydridisation techniques to clones arrayed on a grid, such as cDNAmicroarray hybridisation (Schena et al, Science, 270, 467–470, 1995 andShalon et al, Genome Res, 6, 639–645, 1996) and nucleotide amplificationtechniques such as PCR. A preferred method uses the TAQMAN (Trade mark)technology available from Perkin Elmer. Results from these studies canprovide an indication of the normal function of the polypeptide in theorganism. In addition, comparative studies of the normal expressionpattern of mRNAs with that of mRNAs encoded by an alternative form ofthe same gene (for example, one having an alteration in polypeptidecoding potential or a regulatory mutation) can provide valuable insightsinto the role of the polypeptides of the present invention, or that ofinappropriate expression thereof in disease. Such inappropriateexpression may be of a temporal, spatial or simply quantitative nature.

The polypeptides of the present invention are expressed in brain,kidney, lung, liver.

A further aspect of the present invention relates to antibodies. Thepolypeptides of the invention or their fragments, or cells expressingthem, can be used as immunogens to produce antibodies that areimmunospecific for polypeptides of the present invention. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention maybe obtained by administering the polypeptides or epitope-bearingfragments, or cells to an animal, preferably a non-human animal, usingroutine protocols. For preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohler,G. and Milstein, C., Nature (1975) 256:495–497), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, 77–96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography. Antibodies against polypeptides of the presentinvention may also be employed to treat diseases of the invention,amongst others.

Polypeptides and polynucleotides of the present invention may also beused as vaccines. Accordingly, in a further aspect, the presentinvention relates to a method for inducing an immunological response ina mammal that comprises inoculating the mammal with a polypeptide of thepresent invention, adequate to produce antibody and/or T cell immuneresponse, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said animal from disease, whether thatdisease is already established within the individual or not. Animmunological response in a mammal may also be induced by a methodcomprises delivering a polypeptide of the present invention via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases of the invention.One way of administering the vector is by accelerating it into thedesired cells as a coating on particles or otherwise. Such nucleic acidvector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNAhybrid. For use a vaccine, a polypeptide or a nucleic acid vector willbe normally provided as a vaccine formulation (composition). Theformulation may further comprise a suitable carrier. Since a polypeptidemay be broken down in the stomach, it is preferably administeredparenterally (for instance, subcutaneous, intramuscular, intravenous, orintradermal injection).

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions that may contain anti-oxidants,buffers, bacteriostats and solutes that render the formulation instonicwith the blood of the recipient; and aqueous and non-aqueous sterilesuspensions that may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also include toadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Polypeptides of the present invention have one or more biologicalfunctions that are of relevance in one or more disease states, inparticular the diseases of the invention hereinbefore mentioned. It istherefore useful to to identify compounds that stimulate or inhibit thefunction or level of the polypeptide. Accordingly, in a further aspect,the present invention provides for a method of screening compounds toidentify those that stimulate or inhibit the function or level of thepolypeptide. Such methods identify agonists or antagonists that may beemployed for therapeutic and prophylactic purposes for such diseases ofthe invention as hereinbefore mentioned. Compounds may be identifiedfrom a variety of sources, for example, cells, cell-free preparations,chemical libraries, collections of chemical compounds, and naturalproduct mixtures. Such agonists or antagonists so-identified may benatural or modified substrates, ligands, receptors, enzymes, etc., asthe case may be, of the polypeptide; a structural or functional mimeticthereof (see Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991)) or a small molecule.

The screening method may simply measure the binding of a candidatecompound to the polypeptide, or to cells or membranes bearing thepolypeptide, or a fusion protein thereof, by means of a label directlyor indirectly associated with the candidate compound. Alternatively, thescreening method may involve measuring or detecting (qualitatively orquantitatively) the competitive binding of a candidate compound to thepolypeptide against a labeled competitor (e.g. agonist or antagonist).Further, these screening methods may test whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells bearingthe polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring a GAT2, human GABA transporter2like protein activity in the mixture, and comparing the GAT2, human GABAtransporter2 like protein activity of the mixture to a control mixturewhich contains no candidate compound.

Polypeptides of the present invention may be employed in conventionallow capacity screening methods and also in high-throughput screening(HTS) formats. Such HTS formats include not only the well-establisheduse of 96- and, more recently, 384-well micotiter plates but alsoemerging methods such as the nanowell method described by Schullek etal, Anal Biochem., 246, 20–29, (1997).

Fusion proteins, such as those made from Fc portion and GAT2, human GABAtransporter2 like protein polypeptide, as hereinbefore described, canalso be used for high-throughput screening assays to identifyantagonists for the polypeptide of the present invention (see D. Bennettet al., J Mol Recognition, 8:52–58 (1995); and K. Johanson et al., JBiol Chem, 270(16):9459–9471 (1995)).

Screening Techniques

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents that may inhibit or enhance theproduction of polypeptide (also called antagonist or agonist,respectively) from suitably manipulated cells or tissues.

A polypeptide of the present invention may be used to identify membranebound or soluble receptors, if any, through standard receptor bindingtechniques known in the art. These include, but are not limited to,ligand binding and crosslinking assays in which the polypeptide islabeled with a radioactive isotope (for instance, ¹²⁵I), chemicallymodified (for instance, biotinylated), or fused to a peptide sequencesuitable for detection or purification, and incubated with a source ofthe putative receptor (cells, cell membranes, cell supernatants, tissueextracts, bodily fluids). Other methods include biophysical techniquessuch as surface plasmon resonance and spectroscopy. These screeningmethods may also be used to identify agonists and antagonists of thepolypeptide that compete with the binding of the polypeptide to itsreceptors, if any. Standard methods for conducting such assays are wellunderstood in the art.

Examples of antagonists of polypeptides of the present invention includeantibodies or, in some cases, oligonucleotides or proteins that areclosely related to the ligands, substrates, receptors, enzymes, etc., asthe case may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or a small molecule that bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

Screening methods may also involve the use of transgenic technology andGAT2, human GABA transporter2 like protein gene. The art of constructingtransgenic animals is well established. For example, the GAT2, humanGABA transporter2 like protein gene may be introduced throughmicroinjection into the male pronucleus of fertilized oocytes,retroviral transfer into pre- or post-implantation embryos, or injectionof genetically modified, such as by electroporation, embryonic stemcells into host blastocysts. Particularly useful transgenic animals areso-called “knock-in” animals in which an animal gene is replaced by thehuman equivalent within the genome of that animal. Knock-in transgenicanimals are useful in the drug discovery process, for target validation,where the compound is specific for the human target. Other usefultransgenic animals are so-called “knock-out” animals in which theexpression of the animal ortholog of a polypeptide of the presentinvention and encoded by an endogenous DNA sequence in a cell ispartially or completely annulled. The gene knock-out may be targeted tospecific cells or tissues, may occur only in certain cells or tissues asa consequence of the limitations of the technology, or may occur in all,or substantially all, cells in the animal. Transgenic animal technologyalso offers a whole animal expression-cloning system in which introducedgenes are expressed to give large amounts of polypeptides of the presentinvention

Screening kits for use in the above described methods form a furtheraspect of the present invention. Such screening kits comprise:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the presentinvention;

(c) a cell membrane expressing a polypeptide of the present invention;or

(d) an antibody to a polypeptide of the present invention;

which polypeptide is preferably that of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently hereinbefore.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an

Fab or other immunoglobulin expression library.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide” generally refers to any polyribonucleotide (RNA) orpolydeoxribonucleotide (DNA), which may be unmodified or modified RNA orDNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any polypeptide comprising two or more aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres. “Polypeptide” refers to both short chains,commonly referred to as peptides, oligopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance,Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, 1–12, inPost-translational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York, 1983; Seifter et al., “Analysis forprotein modifications and nonprotein cofactors”, Meth Enzymol, 182,626–646, 1990, and Rattan et al., “Protein Synthesis: Post-translationalModifications and Aging”, Ann NY Acad Sci, 663, 48–62, 1992).

“Fragment” of a polypeptide sequence refers to a polypeptide sequencethat is shorter than the reference sequence but that retains essentiallythe same biological function or activity as the reference polypeptide.“Fragment” of a polynucleotide sequence refers to a polynucloetidesequence that is shorter than the reference sequence of SEQ ID NO:1.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains the essentialproperties thereof. A typical variant of a polynucleotide differs innucleotide sequence from the reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from the referencepolypeptide. Generally, alterations are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, insertions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. Typicalconservative substitutions include Gly, Ala; Val, lie, Leu; Asp, Glu;Asn, Gln; Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of apolynucleotide or polypeptide may be naturally occurring such as anallele, or it may be a variant that is not known to occur naturally.Non-naturally occurring variants of polynucleotides and polypeptides maybe made by mutagenesis techniques or by direct synthesis. Also includedas variants are polypeptides having one or more post-translationalmodifications, for instance glycosylation, phosphorylation, methylation,ADP ribosylation and the like. Embodiments include methylation of theN-terminal amino acid, phosphorylations of serines and threonines andmodification of C-terminal glycines.

“Allele” refers to one of two or more alternative forms of a geneoccuring at a given locus in the genome.

“Polymorphism” refers to a variation in nucleotide sequence (and encodedpolypeptide sequence, if relevant) at a given position in the genomewithin a population.

“Single Nucleotide Polymorphism” (SNP) refers to the occurence ofnucleotide variability at a single nucleotide position in the genome,within a population. An SNP may occur within a gene or within intergenicregions of the genome. SNPs can be assayed using Allele SpecificAmplification (ASA). For the process at least 3 primers are required. Acommon primer is used in reverse complement to the polymorphism beingassayed. This common primer can be between 50 and 1500 bps from thepolymorphic base. The other two (or more) primers are identical to eachother except that the final 3′ base wobbles to match one of the two (ormore) alleles that make up the polymorphism. Two (or more) PCR reactionsare then conducted on sample DNA, each using the common primer and oneof the Alleie Specific Primers.

“Splice Variant” as used herein refers to cDNA molecules produced fromRNA molecules initially transcribed from the same genomic DNA sequencebut which have undergone alternative RNA splicing. Alternative RNAsplicing occurs when a primary RNA transcript undergoes splicing,generally for the removal of introns, which results in the production ofmore than one mRNA molecule each of that may encode different amino acidsequences. The term splice variant also refers to the proteins encodedby the above cDNA molecules.

“Identity” reflects a relationship between two or more polypeptidesequences or two or more polynucleotide sequences, determined bycomparing the sequences. In general, identity refers to an exactnucleotide to nucleotide or amino acid to amino acid correspondence ofthe two polynucleotide or two polypeptide sequences, respectively, overthe length of the sequences being compared.

“% Identity”—For sequences where there is not an exact correspondence, a“% identity” may be determined. In general, the two sequences to becompared are aligned to give a maximum correlation between thesequences. This may include inserting “gaps” in either one or bothsequences, to enhance the degree of alignment. A % identity may bedetermined over the whole length of each of the sequences being compared(so-called global alignment), that is particularly suitable forsequences of the same or very similar length, or over shorter, definedlengths (so-called local alignment), that is more suitable for sequencesof unequal length.

“Similarity” is a further, more sophisticated measure of therelationship between two polypeptide sequences. In general, “similarity”means a comparison between the amino acids of two polypeptide chains, ona residue by residue basis, taking into account not only exactcorrespondences between a between pairs of residues, one from each ofthe sequences being compared (as for identity) but also, where there isnot an exact correspondence, whether, on an evolutionary basis, oneresidue is a likely substitute for the other. This likelihood has anassociated “score” from which the “% similarity” of the two sequencescan then be determined.

Methods for comparing the identity and similarity of two or moresequences are well known in the art. Thus for instance, programsavailable in the Wisconsin Sequence Analysis Package, version 9.1(Devereux J et al, Nucleic Acids Res, 12, 387–395, 1984, available fromGenetics Computer Group, Madison, Wis., USA), for example the programsBESTFIT and GAP, may be used to determine the % identity between twopolynucleotides and the % identity and the % similarity between twopolypeptide sequences. BESTFIT uses the “local homology” algorithm ofSmith and Waterman (J Mol Biol, 147,195–197, 1981, Advances in AppliedMathematics, 2, 482–489, 1981) and finds the best single region ofsimilarity between two sequences. BESTFIT is more suited to comparingtwo polynucleotide or two polypeptide sequences that are dissimilar inlength, the program assuming that the shorter sequence represents aportion of the longer. In comparison, GAP aligns two sequences, findinga “maximum similarity”, according to the algorithm of Neddleman andWunsch (J Mol Biol, 48, 443–453, 1970). GAP is more suited to comparingsequences that are approximately the same length and an alignment isexpected over the entire length. Preferably, the parameters “Gap Weight”and “Length Weight” used in each program are 50 and 3, forpolynucleotide sequences and 12 and 4 for polypeptide sequences,respectively. Preferably, % identities and similarities are determinedwhen the two sequences being compared are optimally aligned.

Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Altschul S F et al, J Mol Biol, 215, 403–410, 1990, Altschul SF et al, Nucleic Acids Res., 25:389–3402, 1997, available from theNational Center for Biotechnology Information (NCBI), Bethesda, Md., USAand accessible through the home page of the NCBI at the world wide webaddress of ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods inEnzymology, 183, 63–99, 1990; Pearson W R and Lipman D J, Proc Nat AcadSci USA, 85, 2444–2448,1988 available as part of the Wisconsin SequenceAnalysis Package).

Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S andHenikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915–10919, 1992) is usedin polypeptide sequence comparisons including where nucleotide sequencesare first translated into amino acid sequences before comparison.

Preferably, the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to areference polynucleotide or a polypeptide sequence, the query and thereference sequence being optimally aligned and the parameters of theprogram set at the default value, as hereinbefore described.

“Identity Index” is a measure of sequence relatedness which may be usedto compare a candidate sequence (polynucleotide or polypeptide) and areference sequence. Thus, for instance, a candidate polynucleotidesequence having, for example, an Identity Index of 0.95 compared to areference polynucleotide sequence is identical to the reference sequenceexcept that the candidate polynucleotide sequence may include on averageup to five differences per each 100 nucleotides of the referencesequence. Such differences are selected from the group consisting of atleast one nucleotide deletion, substitution, including transition andtransversion, or insertion. These differences may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween these terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. In other words, to obtain apolynucleotide sequence having an Identity Index of 0.95 compared to areference polynucleotide sequence, an average of up to 5 in every 100 ofthe nucleotides of the in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, a candidate polypeptide sequence having,for example, an Identity Index of 0.95 compared to a referencepolypeptide sequence is identical to the reference sequence except thatthe polypeptide sequence may include an average of up to fivedifferences per each 100 amino acids of the reference sequence. Suchdifferences are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion. These differences may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between these terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. In other words,to obtain a polypeptide sequence having an Identity Index of 0.95compared to a reference polypeptide sequence, an average of up to 5 inevery 100 of the-amino acids in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

The relationship between the number of nucleotide or amino aciddifferences and the Identity Index may be expressed in the followingequation:n _(a) ≦x _(a)−(x _(a) ∘I)in which:n_(a) is the number of nucleotide or amino acid differences,x_(a) is the total number of nucleotides or amino acids in SEQ ID NO:1or SEQ ID NO:2, respectively,I is the Identity Index,∘ is the symbol for the multiplication operator, andin which any non-integer product of x_(a) and I is rounded down to thenearest integer prior to subtracting it from x_(a).

“Homolog” is a generic term used in the art to indicate a polynucleotideor polypeptide sequence possessing a high degree of sequence relatednessto a reference sequence. Such relatedness may be quantified bydetermining the degree of identity and/or similarity between the twosequences as hereinbefore defined. Falling within this generic term arethe terms “ortholog”, and “paralog”. “Ortholog” refers to apolynucleotide or polypeptide that is the functional equivalent of thepolynucleotide or polypeptide in another species. “Paralog” refers to apolynucleotideor polypeptide that within the same species which isfunctionally similar.

“Fusion protein” refers to a protein encoded by two, unrelated, fusedgenes or fragments thereof. Examples have been disclosed in U.S. Pat.Nos. 5,541,087, 5,726,044. In the case of Fc-human GABA transporter2like protein, employing an immunoglobulin Fc region as a part of afusion protein is advantageous for performing the functional expressionof Fc-human GABA transporter2 like protein or fragments of human GABAtransporter2 like protein, to improve pharmacokinetic properties of sucha fusion protein when used for therapy and to generate a dimericcryptic-like protein. The Fc human GABA transporter2 like DNA constructcomprises in 5′ to 3′ direction, a secretion cassette, i.e. a signalsequence that triggers export from a mammalian cell, DNA encoding animmunoglobulin Fc region fragment, as a fusion partner, and a DNAencoding human GABA transporter2 like protein or fragments thereof. Insome uses it would be desirable to be able to alter the intrinsicfunctional properties (complement binding, Fc-Receptor binding) bymutating the functional Fc sides while leaving the rest of the fusionprotein untouched or delete the Fc part completely after expression.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

1. An isolated polypeptide selected from the group consisting of: (a) apolypeptide encoded by a polynucleotide comprising the sequence of SEQID NO:1; (b) a polypeptide comprising a polypeptide sequence having atleast 95% identity to the polypeptide sequence of SEQ ID NO:2; (c) apolypeptide that is at least 95% identical to the polypeptide sequenceof SEQ ID NO:2; and (d) the polypeptide sequence of SEQ ID NO:2; whereinsaid polypeptide (a)–(d) has GABA transport activity.
 2. The polypeptideof claim 1 comprising the polypeptide sequence of SEQ ID NO:2.
 3. Thepolypeptide of claim 1 which is the polypeptide sequence of SEQ ID NO:2.4. A fusion protein comprising an Immunoglobulin Fc-region and apolypeptide of claim 1.